Modulation ofT cell antigen receptor signaling in CDS+ T lymphocytes following priming with homeostatic and inflammatory cytokines
Written by:
Christopher Lamontagne-Blouin
Department Pediatrics, Division of Immunology
Masters thesis presented to the Faculty of Medicine and Health Sciences in view of obtaining a Master of Science (M.Sc) in Immunology
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Table of Contents
List of Figures IV
List of Abbreviations VIII
Abstract XII
1. Introduction 1
1.1. Selection of CD8+ T cells in the thymus 3
1.2. Activation of CD8+ T cells 7
1.3. The T cell Antigen receptor complex (TCR/CD3) and peptide-MHC 16 interaction
1.4. Signaling events within the CD8+ T cell 21
1.5. Lipid rafts and signaling 30
1.6. Basal homeostatic proliferation of CD8+ T cells 34 1.7. Acute homeostatic proliferation of CD8+ T cells 35 1.8. Antigen-independent proliferation ofCD8+ T cells induced by cytokines 38
leads to astate of 'cytokine priming'
1.9. The yc family of cytokines 43
1.9 .1. IL-7 and the IL-7 receptor (IL-7R) 43
1.9.2. IL-15 and the IL-15 receptor (IL-15R) 44
1.9.3. IL-21 and the IL-21 receptor (IL-21R) 44
2. Hypothesis /Objectives 47
3. Materials and Methods 48
3.1. Animals 48
3.2. Cell lines 48
3.3. Antibodies and reagents 48
3.4. Isolation of lymphocytes 49
3.5. Purification of CD8+T cells subsets 50
3.6. Cytokine priming 50
3.7. Cell activation 51
3.8. Analysis oflipid rafts using cholera toxin B subunit (CTB) 56
3.9. Confocal microscopy 56
3.10. Conjugate formation 60
3.11. Statistical analysis 73
4. Results 76 4.1. Cytokine priming of CD8+ T cells increases LAT phosphorylation after TCR 76
stimulation
4.2. Cytokine priming increases the lipid raft (LR) content and localization of 86 CD45 to LR in naïve CD8+ T cells
4.3. Cytokine priming does not influence the propensity of APC:T cell conjugation 123
5. Discussion 139
6. Acknowledgements 145
List of Figures
Figure 1: Positive and negative selection ofT cells in the thymus. Figure 2: Presentation of intracellular peptides by MHC class I.
Figure 3: Activation of naïve CDS+ T cells: Two signal requirement. Figure 4: Ag-specific CDS+ T cells after acute infections.
Figure 5: Roles of CDS co-receptor in early T cell signaling events. Figure 6: Regulation of Lck activity by CD45.
Figure 7: Barly signaling events in T cells. Figure S: Store-operated calcium entry in T cells.
Figure 9: A model describing the phosphorylation of the ITAM in LR.
Figure 10: Representation of immune cell reconstitution following T lymphopenia. Figure 11. Ag-independent activation of naïve CDS+ T cells by cytokines.
Figure 12. The concept of 'cytokine Priming' ofnaive CDS+ T cells. Figure 13:
Figure 13A. Optimization of TCR stimulation using TCRP-Biotin Ab. Figure 13B. Standardizing the optimal CDSP-Biotin Ab concentration.
Figure 13C. Optimizing the streptavidin concentration for TCR and CDS crosslinking. Figure 13D. Standardizing optimal streptavidin exposure time.
Figure 14. Poly-L-lysine biases the localization of LRs after fixation with low concentration of PFA.
Figure 15:
Figure 15A.
Figure 15A.l. P14 T cells conjugated with peptide-loaded EL4 cells.
Figure 15A.2. Quantitative illustration of the degree of conjugation of P14 CDS+ T cells with GP33 peptide-loaded EL4 cells at different APC:T cell ratios and peptide concentrations.
Figure 15B.
Figure 15B.1. PMEL T cells conjugated with peptide-loaded EL4 ce11s. Figure 15B.2. Quantitative illustration of the degree of conjugation of PMEL
CDS+ T ce11s with MGPlOO peptide-loaded EL4 cells at different APC:T cell ratios and peptide concentrations.
Figure 15C.
Figure 15C.l. NOD S.3 T ce11s conjugated with peptide-loaded RMAS-Kd cells. Figure 15C.2. Quantitative illustration of the degree of conjugation of NOD S.3
CDS+ T cells with NRP-A 7 peptide-loaded RMAS-Kd cells at different APC:T cell ratios and peptide concentrations.
Figure 16. Optimization of conditions for LR isolation by sucrose gradient centrifugation. Figure 17:
Figure 17A. Influence of cytokine priming (IL-7 and IL-7+21) on Tyr phosphorylation events induced by TCR, CDS and TCR+CDS cross-linking in P14 TCR Tg CDS+ T cells.
Figure l 7B. Influence of cytokine priming (lL-7 and IL-7+21) on the phosphorylation of LAT-Y171 induced by TCR, CDS and TCR+CDS cross-linking in P14 TCR Tg CDS+ T cells.
Figure 17C. Influence of cytokine priming (IL-15 and IL-15+21) on Tyr phosphorylation events induced by TCR, CDS and TCR+CDS cross-linking in P14 TCR Tg CDS+ T cells.
Figure 17D. Influence of cytokine priming (IL-15 and IL-15+21) on the phosphorylation of LAT-YI 71 induced by TCR, CDS and TCR+CDS cross-linking in P14 TCR Tg CDS+ T cells.
Figure 17 E. Influence of cytokine priming (IL-7 and IL-7+ 21) on Tyr phosphorylation events induced by TCR, CDS and TCR+CDS cross-linking in S.3 TCR Tg NOD CDS+ T cells.
Figure lS:
Figure ISA.
Figure 18A.1. Quantitative changes in LR content after cytokine priming.
Figure lSA.2. Quantitative illustration of LR surface expression after 4S hours of cytokine priming.
Figure ISE:
Figure 1 SB. J. Kinetics of modulation of LR surface expression after cytokine priming in PMEL TCR Tg CDS+ T cells.
Figure ISB.2. Kinetics of modulation of LR surface expression after cytokine priming in PMEL TCR Tg CDS+ T cells ( continued from the previous figure).
Figure ISB.3. Kinetics of modulation of LR surface expression after cytokine priming in PMEL TCR Tg CDS+ T cells (continued from the previous two figures).
Figure lSC:
Figure 1 SC. J. Kinetics of modulation of LR surface expression after cytokine priming in P14 TCR Tg CDS+ T cells.
Figure lSC.2. Kinetics of modulation of LR surface expression after cytokine priming in P14 TCR Tg CDS+ T cells (continued from the previous figure).
Figure lSC.3. Kinetics of modulation of LR surface expression after cytokine priming in P14 TCR Tg CDS+ T cells (continued from the previous two figures).
Figure lSD:
Figure l SD. I. Kinetics of modulation of LR surface expression after cytokine priming in 8.3-NOD TCR Tg CDS+ T cells.
Figure ISD.2. Kinetics of modulation of LR surface expression after cytokine priming in S.3-NOD TCR Tg CDS+ T cells (continued from the previous figure).
Figure lSD.3. Kinetics of modulation of LR surface expression after cytokine priming in S.3-NOD TCR Tg CDS+ T cells (continued from the
previous two figures). Figure 19:
Figure 19A:
Figure l 9A. l. Cytokine priming promotes colocalization of CD45 with lipid rafts
in P14 cells: Confocal image profile.
Figure l9A.2. Cytokine priming promotes colocalization of CD45 with lipid rafts
in P14 cells: Colocalization graph and intensity profile.
Figure l 9A.3. Cytokine priming promotes colocalization of CD45 with lipid rafts
in P14 cells: Statistical analysis of colocalization. Figure 19B:
Figure l9B.l. Cytokine priming promotes colocalization of CD45 with lipid rafts
in S.3-NOD T cells: Confocal image profile.
Figure l9B.2. Cytokine priming promotes colocalization of CD45 with lipid rafts
in S.3-NOD T cells: Colocalization graph and intensity profile. Figure l9B.3. Cytokine priming promotes colocalization ofCD45 with lipid rafts
in S.3-NOD T cells: Statistical analysis of colocalization. Figure 19C:
Figure 20:
Figure 19C.1. Cytokine priming promotes colocalization of CD45 with lipid rafts in PMEL cells: Confocal image profile.
Figure 19C.2. Cytokine priming promotes colocalization of CD45 with lipid rafts in PMEL cells: Colocalization graph and intensity profile.
Figure 19C.3. Cytokine priming promotes colocalization of CD45 with lipid rafts in PMEL cells: Statistical analysis of colocalization.
Figure 20A:
Figure 20A. l. Effect of cytokine priming on the efficiency of conjugate formation
between P14 T cells and GP33 peptide-loaded EL4 cells.
Figure 20A.2. Quantification of conjugate formation between cytokine-primed
P14 CDS+ T cells and GP33 peptide-loaded EL4 cells.
Figure 20A.3. Effect of cytokine priming on the efficiency of conjugate formation
between P14 T cells and A4Y peptide-loaded EL4 cells.
Figure 20A.4. Quantification of conjugate formation between cytokine-primed
P14 CDS+ T cells andA4Ypeptide-loaded EL4 cells.
Figure 20A.5. Effect of cytokine priming on the efficiency of conjugate formation
between P14 T cells and W4Ypeptide-loaded EL4 cells.
Figure 20A. 6. Quantification of conjugate formation between cytokine-primed P14 CDS+ T cells and W4Ypeptide-loaded EL4 cells.
Figure 20B.
Figure 20B.1. Effect of cytokine priming on the efficiency of conjugate formation
between S.3-NOD T cells and NRP-A 7 peptide-loaded RMAS-Kd cells.
Figure 20B.2. Quantification of conjugate formation between cytokine-primed
List of Abbreviations AA: Ab: ACK: ADAP: Ag: ANOVA: APC: AP-1: a-CPM: p2m: Bcl-2: BMDCs: BSA: Cbp: CD: CRAC: Csk: CTB: CTX: CTL: DAG: DC: DN: DP: DRiP: DRM: ER: Elk: Erk: ETS: FCS: FITC: GEF: GEM: GM-CSF: GMl: GP: Grb2: GTP: yc: HRP: Amino acid Antibody Ammonium chloride
Adhesion and degranulation-promoting Antigen
Analysis of variance Ag-presenting cell Activator protein-1
a-chain connecting peptide motif Beta 2 microglobulin
B cell lymphoma 2
Bone marrow-derived DCs Bovine serum albumin Csk-binding protein Cluster of differentiation Ca2+ -release-activated Ca2+ C-terminal Src kinase CTX B subunit Choiera toxin Cytolytic T lymphocyte Diacylglycerol Dendritic cell Double negative Double positive
Defective ribosomal products Detergent-resistant membranes Endoplasmic reticulum
ETS-like transcription factor 1 Extracellular signal regulated kinase E twenty-six
F etal calf serum
Fluorescein isothiocyanate
Guanine nucleotide exchange factor Glycosphingolipid-enriched domains
Granulocyte-macrophage colony-stimulating factor Monosialotetrahexosy lganglioside
Glycoprotein
Growth factor receptor-bound protein 2 Guanosine triphosphate
Common gamma chain Horseradish perioxidase
ICAM: IFN: Ig: IGRP: IKK: IKB: IL: IL-2R: IL-7R: IL-15R: IL-21R: Ins(l ,4,5)P3: Ins(l,4,5)P3- Rs: IS: ITAMs: Itk: JAK: kDa: LAT: Lck: LCV: LFA-1: LIP: LPS: LR: MAPK: MAPKK: MAPKKK: MFI: MHC: MGPlOO: NFAT: NFKB: NK: NKT: NLR: NOD: NOD: NRP: PAG:
PAMP:
PBS: PDKl:Intercellular adhesion molecule Interferon
Immunoglobulin
Islet-specific glucose 6 phosphatase catalytic subunit related protein IKB kinase Inhibitor of KB Interleukin IL-2 receptor IL-7 receptor IL-15 receptor IL-21 receptor Inositol 1,4,5-triphosphate
Inositol 1,4,5-triphosphate receptors Immune synapse
Immunoreceptor Tyr-based activation motifs IL-2-inducible T cell kinase
Janus kinase Kilodalton
Linker of activated T cells
Lymphocyte-specific protein Tyr kinase Lymphocytic Choreomeningitis Virus Leukocyte function-associated Ag-1 Lymphopenia-induced proliferation Lypopolysaccaride
Lipid raft
Mitogen-activated protein kinase MAPKkinase
MAPKK kinase
Mean fluorescent intensity
Major histocompatibility complex Mouse GPlOO
Nuclear factor of activated T cells
Nuclear factor K-light-chain-enhancer of activated B cells Natural killer
Natural killer T cell NOD-like receptor Non-obese diabetic
Nucleotide binding oligomerisation domain N eurotensin-related peptide
Protein associated with GEM
Pathogen-associated molecular pattern Phosphate buffered saline
PFA: PBK: PIP2: PIP3: PKC: PLCyl: pMHC: PMSF: PRR: Ptdlns(4,5): PVDF: RAG: Rapl: RasGRP: RFI: RIAM: RPMI-FCS-5%: RT: SCID: SDS-PAGE: SFK: SHP-1: SKAP55: SLP-76: Sos: SP: STAT5: STIMl: Syk: TcM: TCA: TCR: TCR/CD3: TEM: Tfh: Th:
TNF:
TLR: TSLP: Tyr: VCAM: Paraformaldehyde Phosphoinositide-3 kinase Phosphatidylinositol ( 4,5) bisphosphate Phosphatidylinositol (3,4,5) trisphosphate Protein kinase C Phospholipase Cyl Peptide:MHC Pheny lmethanesulfonylfluoride Pattern recognition receptorPhosphatidylinositol 4,5 bisphosphate Polyvinylidene difluoride
Recombination-activating gene Ras-proximity-1
Ras guanyl-nucleotide-releasing protein Relative fluorescent intensity
Rapl-GTP-interacting adapter molecule
RPMI-1640, supplemented with 2mM L-glutamine of 1000 IU of penicillin, lOOOµg/ml of streptomycin, lmM of sodium pyruvate, 5.5µM ~-mercaptoethanol and FCS was added to a final concentration of 5% Room temperature
Severe combined immunodeficiency
Sodium dodecyl sulfate polyacrylamide gel electrophoresis Src family kinase
Src homology region 2 domain-containing phosphatase-1 Src kinase-associated phosphoprotein of 55 kDa
SH2 domain-containing leukocyte protein of76 kDa Son of sevenless
Single positive
Signal transducer and activator of transcription 5 Stromal interaction molecule 1
Spleen Tyr
Central memory T cells Trichloroacetic acid T cell receptor
T cell Ag receptor complex Effector memory T cells T follicular helper cells T helper
Tumor necrosis factor Toll-like receptor
Thymie stroma! lymphopoietin Tyrosine
VLA-4: WASp: WAVE: X-SCID: ZAP-70:
Very late Ag-4
Wiskott-Aldrich Syndrome Protein WASp family protein member 2 X-linked SCID
Abstract
Productive stimulation of naïve T cells requires signalling via the T cell antigen receptor (TCR) with concomitant activation of costimulatory receptors. However, naïve CDS+ T cells can undergo antigen-independent proliferation following synergistic stimulation by certain
homeostatic (IL-7 or IL-15) and inflammatory (IL-6 or IL-21) cytokines. These cytokine-stimulated cells proliferate robustly to lower concentrations of antigens or weak TCR agonists, secrete more effector cytokines and display potent antigen-specific cytolytic activity.
Mechanisms underlying the increased antigen sensitivity of such 'cytokine-primed' CDS+ T cells have not yet been elucidated. We used three different TCR transgenic mice bearing the Pl4, PMEL or S.3-NOD TCR on CDS+ T cells to investigate the molecular mechanisms of cytokine priming. Cytokine-primed TCR transgenic CDS+ T cells display an overall increase in tyrosine-phosphorylated proteins following TCR stimulation compared to naïve cells. This increase in protein tyrosine phosphorylation was associated with an increase in CDS expression, and was less pronounced when CDS was also cross-linked along with the TCR. This suggests that cytokine priming may predispose TCR and CDS colocalization, which would enhance phosphorylation of the TCR chains by the Lck kinase associated with CDS. Cytokine-primed CDS+ T cells also displayed increased amounts of plasma membrane lipid rafts, which organize the TCR signalling platform during antigen stimulation. In addition, cytokine priming of CDS+ T cells increased the localization of CD45, a phosphatase that relieves autoinhibition of Lck, in lipid rafts. However, cytokine priming did not affect the ability of CDS+ T cells to form
conjugates with antigen presenting cells pulsed with cognate peptides. Collectively, these
findings suggest that modulation of the composition and functions of lipid rafts may underlie the increased antigen TCR sensitivity of cytokine primed CDS+ T cells.
Résumé
Modulation de la signalisation via TCR chez les lymphocytes T CD8+ suite à une stimulation par cytokines homeostatiques et inflammatoires
La stimulation de cellules T naïves nécessite du déclenchement de la signalisation par l'intermédiaire du récepteur d'antigène de cellule T (TCR) ainsi que l'activation simultanée des récepteurs de co-stimulation. Toutefois, les cellules T CD8+ naïves peuvent proliférer de façon antigène-indépendants suite à la stimulation synergique par certaines cytokines homéostatiques (IL-7 ou IL-15) et inflammatoires (IL-6 ou IL-21 ). Ces cellules pré-stimulées prolifèrent même à des faibles concentrations d'antigènes ou en presence d'agonistes du TCR. Ceci leur permet de sécréter des cytokines effectrices, d'être plus spécifiques à leur antigène et d'avoir une activité cytolytique plus importante. Les mécanismes déclenchés par les cellules T CD8+ permettant une sensibilité accrue à l'antigène suite à la «pré-stimulation aux cytokines» n'ont pas encore été élucidés. Nous avons utilisé trois différents modèles de souris transgéniques portant le TCR Pl4, PMEL ou 8.3-NOD sur les lymphocytes T CD8+ afin d'étudier les mécanismes moléculaires suite à la pré-stimulation aux cytokines. Les cellules T CD8+ portant le TCR transgénique amorcées avec les cytokines, possèdent une augmentation globale des protéines
tyrosine-phosphorylés après stimulation du TCR par rapport aux cellules naïves. Cette augmentation de la phosphorylation de la protéine tyrosine a été associée à une augmentation de l'expression de CD8, et a été moins prononcé lorsque CD8 a également été réticulés avec le TCR. Ceci suggère que l'amorçage aux cytokines peut prédisposer le TCR et CD8 à colocaliser, ce qui renforcerait la phosphorylation des chaînes du TCR par la kinase Lck associée à CD8. Les lymphocytes T CD8+ amorcées aux cytokines présentent également des quantités accrues de radeaux lipidiques plasmatiques à la membrane, qui organisent la plate-forme de signalisation du TCR au cours de la stimulation antigénique. L'amorçage aux cytokines des lymphocytes T CD8+ a également augmenté la localisation de CD45, une phosphatase qui diminue l'inhibition automatique de la Lck dans les radeaux lipidiques. Cependant, l'amorçage aux cytokines n'a pas d'incidence sur la capacité des cellules CD8+ T pour former des conjugués avec les cellules présentatrices
d'antigène puisées avec des peptides apparentés. En conclusion, ces résultats suggèrent que la composition et les fonctions des radeaux lipidiques peuvent moduler la sensibilité à l'antigène via le TCR lorsque les lymphocytes T CD8+ ont été pré-stimulés aux cytokines.
Mots Clés: cellules T CD8+, IL-7, IL-15, IL-21, cytokine priming, CD8, Lck, radeaux lipidiques, CD45
1. Introduction
Mature cytolytic T lymphocytes (CTL) expressing the CDS (Cluster of differentiation S) cell surface molecule are an important arsenal of the adaptive immune system that play crucial roles in the defense against viroses, intracellular pathogens and cancer cells (Harty 2000). In order to exert their cytolytic functions, CDS+ T cells must become activated through their antigen (Ag) receptors, known as T cell receptors (TCRs). TCRs on CDS+ T cells recognize Ag presented by the class 1 major histocompatibility complex molecules (MHC-1) expressed on Ag-presenting cells (APCs) such as dendritic cells (DCs) and macrophages, which also provide the obligatory co-stimulatory signais. Simultaneous signaling via the TCR and the costimulatory receptors leads to robust proliferation of CDS+ T cells and their differentiation into cytolytic T lymphocytes (CTLs) (Banchereau 199S). This process occurs in draining lymph nodes and is facilitated by the help provided by CD4+ T cells. The effector functions of CTLs manifest against pathogen-infected cells, cancer cells and, occasionally, healthy cells in situations of autoimmune disease (Harty 2000). Upon elimination of the infection, excess CDS+ T cells undergo a contraction phase which eliminates 90% to 95% of activated cells. Roughly 5% of these activated cells differentiate into memory CDS+ T cells, with the help of interleukin (IL)-15, and confer long-term immunity (Murali-Krishna 1999).
Under certain conditions, such as conditions of lymphopenia, naïve CDS+ T cells can undergo Ag-independent proliferation in order to restore the T cell pool. This process is referred to as lymphopenia-induced proliferation (LIP) and is driven by an increased availability of cytokines, specifically IL-7 and IL-15 (Jameson 2002). Interestingly, it has been shown that certain proinflammatory cytokines, such as IL-21, can synergize with IL-7 or IL-15 to enhance
proliferation further. Specifically, previous work in our laboratory as well as others bas shown that this synergy can induce Ag-independent proliferation of CD8+ T cells and can also enhance the Ag responsiveness ofnaive CD8+ T cells (Zeng 2005; Gagnon 2007; Gagnon 2008; Gagnon 2010). In my project I investigated the mechanisms by which the synergistic combination of cytokines enhance the sensitivity of naïve CDS+ T cells.
1.1. Selection of CDS+ T cells in the thymus
T lymphocytes undergo maturation in the thymus. Progenitor T cells, derived from bone marrow cells, migrate to the thymus with the help various chemokines {Takahama 2006) and develop into different lineages: ap or
yo
TCR-expressing T cells, Natural Killer (NK.) cells and NK T cells. Thymie development of progenitor cells into T cells expressing the ap TCR is a multistep process characterized by the presence or absence of the CD4 or CDS markers: the double negative (DN), double positive (DP) and single positive (SP) stages (Zuniga-Pflucker 1996) (Fig. 1 ).Committed lymphoid precursors, which migrate from the bone marrow to the thymus lose their potential for B cell and NK development and begin their lives as DN (CD4-CDS-) thymocytes. The DN stage is subdivided into four developmental stages in which CD44 and CD25 are differentially expressed. DN3 thymocytes that are CD44-CD25+ express the pre-TCRa encoded by a non-rearranging locus. These cells begin to express TCRP through somatic DNA rearrangements requiring the recombination-activating gene (RAG) 1 and 2 proteins. Failure to rearrange the TCRP chain leads to apoptosis (Zuniga-Pflucker 2004). Cells that have successfully rearranged the TCRP gene signal via the pre-TCRa:TCRP complex, leading to rearrangement of the TCRa locus, leading to the formation of the mature ap Ag receptor and the expression ofboth CD4 and CDS molecules (von Boehmer 1997; Germain 2002). Cells that fail to rearrange the TCRa locus undergo death while the CD4+CDS+ double positive (DP)
thymocytes proceed to the next stage of thymie positive selection. During this process, the TCRap ofDP thymocytes recognize, with low affinity, self-peptides expressed on MHC molecules of thymie epithelial cells. If the recognized peptide is expressed on MHC class I
(MHC-1) molecules, the DP cells differentiate into CDS SP thymocytes, whereas recognition of a peptide presented on MHC class II (MHC-11) molecules promotes maturation towards CD4 SP thymocytes. DP thymocytes possessing TCRs which fail to recognize any peptide:MHC complexes undergo death by neglect (Goldrath 1999; Sebzda 1999).
On the other hand, DP thymocytes that express TCRs which recognize the
self-peptide:MHC molecules with high affmity undergo the negative selection process in the cortex and, at the cortico-medullary junction, undergo death. This negative selection process constitutes the central tolerance mechanism to eliminate potentially autoreactive T cells (Goldrath 1999; Sebzda 1999; Takahama 2006). Four-to-five days after becoming SP thymocytes, these naïve a~ T cells emigrate to the periphery where they undergo further maturation as naïve T cells
(Takahama 2006; McCaughtry 2007). Mature SP T cells then migrate from the thymus into the periphery and populate the secondary lymphoid organs. These cells circulate throughout the body in search ofTCR-specific Ag expressed on professional Ag presenting cells (Abbas 2006;
Murphy 2011 ).
In the periphery, naïve T cells live for a long time even in the absence of any Ag stimulation, but rel y on two key homeostatic signais for prolonged survival. One of these homeostatic eues is IL-7, which engages the IL-7 receptor (IL-7R) on naïve T cells and induces anti-apoptotic proteins (Aspinall 2006; Jacobs 2010). Second, naïve T cells require a basal level of TCR signaling provided by transient engagement of self-peptide:MHC complexes (Brown 2005).
'- X a.> Ql ... t ::J 0 0 u '- X Ql Ql c:: t c:: 0 - u
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Figure 1: Positive and negative selection of T cells in the thymus.
recursor
Mature bone marrow stem cells that become lymphoid progenitors rnigrate to the thymus. Those destined to become T cells begin their development as DN thymocytes (TCR-CD4-CD8-). There are four stages of differentiation of DN thymocytes based on their expression of CD25 and CD44, designated DN1-DN4: DNl, CD44+CD25-; DN2, CD44+CD25+; DN3, CD44-CD25+; and DN4, CD44-CD25-. Rearrangement of the TCRP chain occurs during this stage and is dependent on IL-7. The DP stage is characterized by the rearrangement of the a chain of the TCR. During this stage these cells undergo positive selection; a process in which DP cells
become SP T cells ( either CD4+ or CDS+) depending on whether their TCR recognizes peptides expressed on MHC-II or MHC-1, respectively. If the affinity of the TCRs ofthese T cells to the MHC:self-peptide is too weak, they die by neglect. If the TCRs have too high an affinity towards self-peptide:MHC complex (pMHC), the cells die by apoptosis. The survival of the newly developed SP T cells depends on IL-7 [Illustration modified from (Germain 2002)].
1.2. Activation of CDS+ T cells
The first line of defense against invading pathogens are cells of the innate immune system. Infectious agents that are able to penetrate the epithelial barri ers of the body (the
epidermis, gastrointestinal tract or respiratory tract) may corne into contact with sentine! cells of DC and macrophage lineages that function as professional APCs (Abbas 2006; Murphy 2011 ). Epithelial DCs are unable to stimulate T cells and are considered immature. Upon recognizing pathogen-associated molecular patterns (PAMPs) residues expressed by pathogens, which are usually necessary for their survival, through their pattern recognition receptors (PRRs ), such as Toll-like receptors (TLRs) and nucleotide binding oligomerisation domain (NOD)-like receptors (NLRs), they become activated. Activated DCs can endocytose microbial pathogens, process and presentAg and upregulate costimulatory molecules (Banchereau 1998). Once activated, DCs migrate to draining lymph-nodes due to the upregulation of certain chemokine receptors. Activated DCs upregulate MHC molecules as well as costimulatory ligands, which promotes their functionality as professional APCs (Banchereau 1998; Abbas 2006; Murphy 2011).
Peptides loaded onto MHC class I and II molecules are processed differently. Ag peptides presented by MHC class II molecules are generated by endosomal proteases, which degrade the proteins of endocytosed pathogens. The peptides are subsequently loaded onto MHC class II molecules and are transported to the cell surface where they may be recognized by the TCRs of CD4+ T cells (Neefjes 2011 ). The Ag peptide presented by MHC class I molecules are generated by cytosolic proteases, as well as the proteasome and immunoproteasome, which leads to the degradation of cytosolic microbial proteins and defective ribosomal products (DRiPs). These peptides are then transported from the cytosol into the endoplasmic reticulum (ER) with the help
of a traffic ATPase known as the transporter associated with Ag processing (TAP). Here these peptides are assembled into complexes with nascent MHC class I molecules and beta 2 microglobulin (P2m) and are eventually transported to the cell membrane where they may be recognized by the TCRs of CDS+ T cells (Cresswell 2000; Van Kaer 2002; Neefjes 2011) (Fig. 2).
Upon encountering DCs, naïve CDS+ T cells require two signals in order to become activated: TCR-peptide:MHC (pMHC) ligation and costimulation (Fig. 3). TCR-pMHC ligation arises from the interaction of a TCR expressed by naïve CDS+ T cells with its specific antigenic peptide presented by MHC class I molecules and expressed by professional APCs. This
interaction is facilitated by the interaction of the CDS coreceptor with the MHC class I molecule (Kerry 2003). The second signal arises from costimulatory receptors. During the maturation of DCs into professional APCs, which occurs during their migration to draining lymph nodes, these cells upregulate costimulatory ligands such as CDSO or CDS6. The ligands are recognized by the costimulatory receptor CD2S, which is expressed by CDS+ T cells (Banchereau 199S; Acuto 2003; Abbas 2006; Murphy 2011). Naïve CDS+ T cells require a few hours in order to be fully stimulated by DCs (van Stipdonk 2001 ). In the absence of costimulation, unless the affmity of the TCR with the pMHC is very high, the TCR becomes unresponsive to subsequent antigenic stimulation in a process known as anergy (Schwarts 2003).
Aside from signals 1 and 2 it has been shown that various inflammatory cytokines,
particularly IL-12, the type 1 interferons (IFN) a and
p,
as well as IFNy, also help to enhance the activation of CDS+ T cells (Haring 2006). This is referred to as signal 3 and it has been shownthat naive CD8+ T cells which have undergone Ag-stimulation require this signal for efficient clonal expansion, survival, and acquisition of full effector functions (Haring 2006) (Fig. 3).
Activated CD8+ T cells undergo clonal expansion under the influence of IL-2, a paracrine and autocrine growth factor secreted by activated CD4+ and CD8+ T cells that also promotes CD8+ T cell differentiation into effector cells (D'Souza 2001). Besides IL-2, IFNy secreted by activated T cells promote CD8+ T cell differentiation towards cytolytic T lymphocytes (CTLs). CTLs enter the circulation in search of other infected cells. Upon encountering target cells, CTLs release granzyme Band perforin which induce cytolysis (Harty 2000). In addition to this, CTLs also induce apoptosis of target cells via Fas ligand, which interacts with its receptor located on target cells.
Around 90-95% of CTLs die following the elimination of pathogen during the contraction phase of the immune response. This reduction in the number of effector CD8+ T cells occurs due to the absence of pathogen, which causes reduced signaling through the TCR, as well as the increased competition for survival cytokines. Those cells that remain, roughly 5%, become memory T cell and confer long-term immunity (Badovinac 2006). Two types of memory cells are generated following an immune response: effector memory (T EM) and central memory (T cM) T cells. T EM cells reside in the peripheral tissues, are characterized by the CCR 710, CD62L1° and IL-7Rhi phenotype and are able to immediately become CTLs and induce cytolytic activity upon recognition of the sameAg. TcMcells on the other hand are long-lived cells, which circulate through lymph nodes and are characterized by CCR7hi and CD62Lhi and IL-7Rhi phenotype. These cells are able to proliferate rapidly and produce more effector cells in response to recognition of the same Ag (Seder 2003; Badovinac 2006). ). IL-7 and IL-15 are both required
for the survival of memory CD8+ T cells (Lodolce 1998; Kennedy 2000; Sallusto 2004; Mazzucchelli 2007) (Fig. 4).
TCR
0
Peptide MHC Class 1 B2m Golgi ER0
Proteins in cytosol or nucleus\o
Dt:!
ProteasomeThe presentation of intracellular Ags, either pathogenic or derived from DRiPs, by MHC class 1 molecules is a multistep process. Ags are first cleaved by the proteasome into peptides of eight or nine AAs in length. Peptides are then transported into the ER lumen via TAP and are loaded on MHC class 1 molecules. From here, the peptide-MHC class 1:~2m complexes are shuttled to the plasma membrane via the Golgi in a complex. Ags presented by MHC class 1 molecules are recognized by the Ag-specific TCRs of CDS+ T cells [Illustration modified from (Neefjes 2011)].
IL-2
IL-7
APC.
Signal 1IU.l,Fid
J
@
CTLs Pathogens1 1
1 1
lnnate immune stimulation
IL-15
Clonal expansion
Memorycell
Figure 3: Activation of naïve CD8+ T cells: Two signal requirement.
ln order to become fully activated naïve CD8+ T cells requires two essential signais: activation through the TCR and co-stimulation. Recognition of TCR-specific peptides presented by MHC-I
molecules, expressed by APCs, by the TCRs of naïve CDS+ T cells constitutes the first signal. The second signal, costimulation, arises through the ligation of the costimulatory ligand ( such as CDSO or CDS6) expressed by APCs with CD2S of T cells. Activated T cells undergo clonal expansion under the influence of IL-2, which can be substituted by IL-7 or IL-15. In addition to signal one and two, the activity of certain pro-inflammatory cytokines, such as IL-12 and IFNa, can boost the efficiency of the CDS+ T cell response. Once activated, CDS+ T cells become CTLs. The se CTLs die after elimination of the pathogen. A small proportion of activated cells become memory cells, which provide long-term immunity. IL-15 sustains the survival and renewal ofmemory CDS+ T cells [Illustration modified from (Ramanathan 2009)].
"'
ai u 1-co 0 u '+-0 =1:1:: ClJ > ·.;:; ro ClJ a:: CD127 CD62L CCR7 IFN-y IL-2 Na ive Effectorî
Hi Lo Hi Lo Hi Lo Lo Hi Lo LoFigure 4: Ag-specific CD8+ T cells after acute infections.
Memory LOD Ti me Hi Lo&Hi Lo& Hi Hi Lo& Hi
Naïve CD8+ T cells are characterized by a CD127hi, CD62Lhi and CCR7hi phenotype. After infection, CD8+ T cells undergo robust proliferation during the expansion phase, which is characterized by high expression ofIFNy. Most of these cells die during the contraction phase except for roughly 5% of activated CD8+ T cells, which differentiate into memory cells. Often, the memory CD8+ T-cell pool will be comprised of multiple subpopulations with distinct functional characteristics. LOD represents the limit of detection [Illustration modified from (Badovinac 2006)].
1.3. The T cell Antigen receptor complex (TCR/CD3) and peptide-MHC interaction
The TCR/CD3 is the cell surface receptor responsible for Ag recognition and the triggering of signals needed to mount adequate responses necessary to eliminate foreign pathogens. It is composed of three chains. The TCR chain is responsible for the detection of foreign Ag presented in the context of self-MHC molecules, and the invariant CD3 (CD3E,
CD3y, CD3o) and CD247 (Ç) chains that transduce the signal (Roja 2008).
The TCR is composed of two disulfide-linked class I membrane glycosylated polypeptides named a and p. Each chain contains one variable (V) and one constant (C)
immunoglobulin (Ig) domain linked by a disulphide bridge. The ap TCR specifically recognizes a short antigenic peptide presented by the MHC molecules (Clevers 1988; Kuhns 2006; Rudolph 2006). The TCR has no enzymatic activity of its own and requires the CD3 chains, specifically
the CD3y, CD3o and two CD3E chains, as well as two Ç chains to convey the signal of ligand
binding by the TCR within the cell. On the cell surface, the TCR is non-covalently associated
with the invariant CD3 E, y and o polypeptides and the Ç homodimer. The CD3 and Ç chains
possess relatively large intracellular domains, which contain immunoreceptor Tyr-based
activation motifs (ITAMs). Each CD3 subunit possesses one ITAM motif and each Ç subunit
possesses three (Rudolph 2006; Roja 2008). These ITAMs become phosphorylated during TCR engagement ofpMHC and the phospho-ITAM motifs propagate the cellular activation signal.
The MHC class-I and class-II molecules have similar architecture. Bath are heterodimers and are composed of two chains; the peptide-binding site composed of one a-helix/P-sheet ( ap)
superdomain, and two Ig-like domains. In class I MHC molecules, which associate with the
only. This heavy chain is referred to as the ala2 domain and contains a groove, which can house the peptide Ag and present it (Madden 1993; Rudolph 2006). Only two to five peptide residues presented in this way are recognizable by the TCR (Garboczi 1996; Garcia 1996). The peptides that bulge most out of the groove are referred to as functional hotspots in the TCR/pMHC synapse (Degano 2000; Rudolph 2002). This polymorphie heavy chain associates with the light chain subunit P2m (Madden 1993; Rudolph 2006).
T cells possessing TCRs that recognize peptides bound to MHC class-1 molecules express the CDSap coreceptor. This coreceptor, as well as the CD4 coreceptor expressed on T cells that recognize peptides presented by MHC-II, are associated with lymphocyte-specific protein tyrosine (Tyr) kinase (Lck), which phosphorylates the CD3 and Ç ITAM motifs during TCR signaling (Veillette 19SS; Baniyash 2004). CDS binds MHC class-1 molecules via interactions with largely nonpolymorphic amino acid residues situated in the a3 and a2 domain of the polymorphie heavy-chain and P2m (Lau gel 2011 ). Through this interaction, CDS increases the association rate and enhances the half-life interaction of pMHC:TCR ligation (Gakamski 2005; Wooldridge 2005). Specifically, palmitoylation of the CDSP-chain enables it to internet directly with CD38 (Laugel 2011 ). CDS facilitates the recruitment of TCR/CD3 complexes to membrane microdomains wherein spatial segregation of the TCR/CD3 complex from inhibitory
phosphatase proteins, particularly CD45, promotes TCR signaling. In addition, the TCR a-chain connecting peptide motif ( a-CPM) supports the recruitment of CDS in close proximity to the TCR/CD3 complex via unknown mechanisms (Naeher 2002; Mallaun 200S). This colocalization of this coreceptor with TCR/CD3 presumably facilitates Lck Tyr kinase phosphorylation of ITAMs. Meanwhile, CDS interaction with class I MHC promotes pMHC:TCR ligation, which
acts to enhance T cell affinity towards the peptide Ag. It is important to note that CD8
recruitment to TCR/CD3 requires prior TCR engagement implying that :free Lck phosphorylation o:flTAMs is a necessary precursor (Laugel 2011) (Fig. 5).
TCR
(03
Step 1Q
Zap70
p
CD8
Rafts and
associated
molecules
....
~Directmolecular interaction
--->...;:::i•
Kinase activity
T cell activation
Figure 5: Roles of CDS coreceptor in early T cell signaling events.
TCR engagement to pMHC initiates a series of events, which promotes CDS recruitment to
TCRJCD3 complexes. CDS is able to recroit TCRJCD3 complexes to membrane microdomains
due to the palmitoylation of the CDSP-chain, which enable this chain to interact directly with CD38. Stimulation through the TCR leads to a small degree of Tyr phosphorylation events of CD3 and Ç ITAM motifs presumably due to the kinase activity of free Lck. This limited
phosphorylation is necessary for the recruitment of CD8 within close proximity to the TCR/CD3 complex, due to unknown mechanisms, which require TCRa-CPM. CD8 is then able to interact with class-1 MHC, which enhances the association and half-life of the pMHC:TCR interaction [Illustration modified from (Laugel 2011)].
1.4. Signaling events within the CDS+ T cell
Upon engagement of the TCR by specific pMHC a number of signaling events take place. The Src family kinase (SFK) Lck becomes activated due to a relief in inhibition mediated by the phosphatase CD45. Essentially, Lck activity is largely regulated by the phosphorylation status activity oftwo key Tyr residues: one in the kinase domain (residue 394) and one at the C-terminus (residue 505). Phosphorylation at th~ kinase domain enhances kinase activity, whereas phosphorylation at the C-terminal Tyr is inhibitory. The phosphatase activity of CD45 is capable ofremoving the phosphates from both ofthese residues enabling CD45 to both enhance and diminish the activity of Lck. Following TCR ligation, CD45 removes the phosphate from the inhibitory Tyr ofLck (McNeill 2007; Zamoyska 2007) (Fig. 6). Once activated, Lck
phosphorylates the ITAMs of the Ç homodimer, which promotes the recruitment and activation of the Ç-associated protein of 70 kilodaltons (kDa) (ZAP-70), another Tyr kinase. ZAP-70 then phosphorylates the linker of activated T cells (LAT) and SH2 domain-containing leukocyte protein of76 kDa (SLP-76) (Wardenburg 1996; Zhang 1998). LAT and SLP-76 are adaptor proteins and their phosphorylation leads to the recruitment of a number of other proteins involved in calcium mobilization, the activation of the Ras pathway and cytoskeletal rearrangement (Iwashima 1994; Lin 2001) (Fig. 7).
One protein which is recruited to LAT after its phosphorylation is phospholipase Cyl {PLCyl). This protein cleaves phosphatidylinositol 4,5 bisphosphate [Ptdlns(4,5)P2] at the plasma membrane into diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins{l,4,5)P3] (Beach 2006). DAG is responsible for activating a number of proteins; notably various isoforms of protein kinase C (PKC) and Ras guanyl-nucleotide-releasing protein (RasGRP), whereas Ins
(1,4,5)P3 plays a critical role in calcium mobilization. Ins(l ,4,5)P3 binds to Ins(l ,4,5)P3feceptors
[Ins(l,4,5)P3- Rs] on the surface of the ER, which initiates the release of Ca2+ stores into the
cytoplasm. The decrease in ER stores of Ca2+ is detected by stromal interaction molecule 1
(STIMl), which in turn triggers the release of Ca2+-release-activated Ca2+ (CRAC) channels at
the plasma membrane causing an influx of extracellular Ca2+. This prevents calmodulin
inhibition of the protein phosphatase calcineurin. Activated calcineurin then dephosphorylates nuclear factor of activated T cells (NFAT). NFAT is a transcription factor which, once
dephosphorylated, translocates to the nucleus where it is able to work with other transcription factors to bind promotors (Lin 2001; Feske 2007; Oh-hora 2008) (Fig. 8).
Recruitment of the Ras exchange factors, son of sevenless (Sos) and RasGRP, to the cell membrane is required for its activation. RasGRP is inducibly recruited to the cell membrane through a DAG-binding domain and promotes the formation of Ras guanosine triphosphate (GTP) (Stone 2011). Ras-GTP promotes the activation of serine/threonine kinases and dual-specificity kinases that are responsible for the eventual activation of mitogen-activated protein kinases (MAPKs) extracellular signal regulated kinase (Erk) 1 and 2, c-Jun N-terminal kinases (JNK) and p38. These MAPKs phosphorylate transcription factors involved in the formation of the transcription factor activator protein-1 (AP-1 ), among others (Lin 2001 ). An example of this is the serine/threonine kinase Rafl. Rafl is a MAPK kinase kinase (MAPKKK), which
phosphorylates and activates MAPK kinases (MAPKKs), which in turn phosphorylate and
activate the MAPKs Erkl/2. Erk kinase activity results in the activation of E twenty-six (ETS)-like transcription factor 1 (Elkl), which contributes to AP-1 activation (Genot 2000; Smith-Garvin 2009).
In addition to the activation of transcription factors that induce genes regulating cytokine production, cell proliferation and cell differentiation, TCR-pMHC interaction also leads to a program of actin cytoskeletal rearrangements that results in polarization and activation of the T cell (Smith-Garvin 2009). Activation of the Lck and ZAP-70 kinases following TCR signaling leads to the formation of a signaling complex at the immune synapse (IS), the area of contact between the TCRs of T cells and the pMHC of APCs. This signaling platform beneath the plasma membrane contains the adaptor proteins LAT and SLP-76, the Tee family kinase IL-2-inducible T cell kinase (Itk), and the Rho family guanine nucleotide exchange factor (GEF) Vavl
(Burkhardt 2008). This stable signalosome complex, also containing other signaling proteins, promotes actin polymerization via Wiskott-Aldrich Syndrome Protein (WASp) and WASp family protein member 2 (WAVE2) (Labno 2003; Dombroski 2005).
Formation of the IS requires the activity ofvarious integrins, a~ heterdimeric receptors which form a physical link between T cells and APCs. Important T cell integrins include:
leukocyte function-associatedAg-1 (LFA-1) and very lateAg-4 (VLA-4). These integrins bind to their ligands, mainly intercellular adhesion molecule (ICAM), and vascular cell adhesion
molecule (VCAM) and fibronectin, respectively. These ligands are expressed by other immune cells, epithelial cells, fibroblasts and extracellular matrix proteins (Smith-Garvin 2009). The expression of integrins and their affinity for their ligand is controlled by a number of intracellular proteins such as Ras-proximity-1 (Rap 1 ), which requires TCR activation and the activity of the membrane-proximal proteins LAT, SLP-76 and PLCyl for its activation. Rapl requires the formation of the adhesion and degranulation-promoting (ADAP) adaptor protein/Src kinase--associated phosphoprotein of 55 kDa (SKAP55)/Rap 1-GTP-interacting adapter molecule
(RIAM) complex for its proper localization near the cell membrane. The impact of intracellular signaling on integrin expression is a process referred to as 'inside-out signaling' (Menasche 2007a; Menasche 2007b).
This section has thus far examined the impact ofTCR activation on intracellular signaling and the consequences of various protein interactions. lt was mentioned before that ligation through the TCR alone is insufficient to fully activate T cells and can lead to anergy (Schwarts 2003). To escape anergy, activated T cells must receive a co-stimulatory signal via costimulatory receptors such as CD28. One consequence of CD28 ligation is that it leads to the conversion of phosphatidylinositol (4,5) bisphosphate (PIP2) to phosphatidylinositol (3,4,5) trisphosphate (PlP3) at the membrane. This membrane-proximal PlP3 serves as a docking site for 3-phospoinositide-dependent protein kinase 1 (PDKl) and its target Ak.t. Ak.t phosphorylates multiple proteins and has an impact on many cellular responses (Smith-Garvin 2009). One example is the role of Ak.t on the transcription of nuclear factor K-light-chain-enhancer of activated B cells (NF-KB). NF-KB is a transcription factor important for the generation ofIL-2. NFKB is dependent on TCR ligation and costimulation via CD28 (Acuto 2003). Essentially, NFKB is regulated by the inhibitor of KB (IKB) kinase (IKK), which phosphorylates IKB, leading to ubiquitination. After inhibition of IKB on NFKB is relinquished, NFKB can move into the nucleus to activate transcription. Activation of the IKB complex is performed by the serine/ threonine kinase Akt and the MAPKKKs, which are activated via the costimulatory signaling pathway (Thompson 1995; Ballard 2001; Lin 2001; Cheng 2011 ). Costimulatory signaling also plays a role in actin cytoskeletal rearrangement due to phosphoinositide-3 kinase (PBK)-dependent activation and localization ofVavl and ltk (Burkhardt 2008). Essentially, CD28
promotes T cell proliferation, cytokine production (via gene transcription and mRNA stability), cell survival, and cellular metabolism (Acuto 2003).
lt is important to note that T cell signaling is regulated at all levels. One example was detailed above in which C-terminal Src kinase (Csk) phosphorylates Lck on its inhibitory Tyr residue (Y505), which is countered by the phosphatase activity of CD45 (Hermiston 2003; Zamoyska 2007). Another important regulator is CD5. Evidence suggests that CD5 may internet with the TCR-p23 Ç chains, thus preventing ZAP-70 binding (Gary-Gouy 1997). In order to accomplish this, it is possible that after TCR engagement with Ag, the phosphorylation of serine residues on the CD5 cytoplasmic tail may act as a docking site for a Tyr phosphatase such as Src homology region 2 domain-containing phosphatase-1 (SHP-1 ). This would in turn
dephosphorylate the Ç chain of the ITAMs, thus inhibiting the recruitment of ZAP-70 (Davies 1992; Dalloul 2008).
CD45
CD45
p
Ysos
CD45
Lck
Csk
CD45
Y394 Y394Ysos
Yso
Inactive Lck Basally active Lck Fully active Lck
Figure 6: Regulation of Lck activity by CD45.
The inhibitory Tyr of Lck (Y505) is phosphorylated by the kinase Csk. This causes Lck to adopt an inactive conformation. The phosphatase activity of CD45 removes this inhibition causing Lck to return to its basally active state. After TCR ligation to Ag, this basally active pool of Lck becomes phosphorylated on its activating Tyr (Y394) causing Lck to adopt its fully active conformation. Similarly, the phosphatase activity of CD45 dephosphorylates this Tyr as well to maintain a pool of basally active Lck [Illustration modified from (Zamoyska 2007)].
Costimulation CO N 0 u LAT
,
SLP76 pMHCI Calcium mobilization Activation of RAS pathways Cytoskeletal rearrangementFigure 7: Barly signaling events in T cells.
(X)
8
•
®
'
8
Ligation of the TCR with pMHC leads to a complex cascade of intracellular signaling events. TCR engagement leads to Lck phosphorylation of the ITAMs, which recruit ZAP-70. ZAP-70 phosphorylates the linker proteins LAT and SLP-76, which orchestrate signaling events leading to calcium mobilization, the activation of the Ras pathway and cytoskeletal rearrangement. These
processes, complemented by signaling via CD28 and other costimulatory receptors, eventually culminate in the activation of a number of transcription factors that regulate cytokine secretion and cytolytic activity in the case of CDS+ T cells [Illustration modified from (Lin 2001 )].
Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ ER
Figure 8: Store-operated calcium entry in T cells.
Ca2+
hannel Plasma membrane
@
<Q"MKll/CaM@J> 1 ~i
ti
@
Cytoplasm NucleusIn resting T cells there is a large gradient between the levels of cytosolic Ca2+ and those of the
lumen of the ER. TCR activation fundamentally leads to the catalysis of Ptdlns(4,5)P2 into DAO and Ins(l ,4,5)P3. Ins(l ,4,5)P3 binds toits receptor located on the ER, which causes a release of Ca2+ from the lumen of the ER into the cytoplasm. The decrease in ER Ca2+ is detected by STIM, which triggers the release of CRAC channels at the plasma membrane. This causes an influx of Ca2+, which causes a drastic increase in the levels of cytoplasrnic Ca2+, which in tum relieves
calmodulin inhibition of calcineurin. Activated calcineurin then dephosphorylates NFAT, which translocates to the nucleus to initiate gene transcription [Illustration modified from (Feske 2007)].
1.5. Lipid rafts and signaling
TCR signaling involves a complex cascade of signaling events and temporally orchestrated signaling events. Following TCR ligation, Lck is brought to the CD3:TCR complex by the co-receptor CD8. Besides, free-Lck residing within lipid rafts (LR) at the immune synapse is relieved in inhibition by the phosphatase activity of CD45 (Fooksman 2010). Activated Lck phosphorylates the ITAM motifs on TCRÇ chains, which recuit ZAP-70 to be phosphorylated and activated by Lck. Activated ZAP-70 subsequently phosphorylates LAT and SLP-76, initiating a complex signaling cascade detailed in the previous section (Iwashima 1994; Wardenburg 1996; Zhang 1998; Lin 2001; McNeill 2007; Zamoyska 2007).
Over the past decade, intensive efforts have been made to understand how LR influence/ modulate the TCR signaling. LRs are defined as membrane microdomains enriched in
cholesterol, glycosphingolipids and sphingomyelin, which differ from the glycerophospholipid bilayer that makes up the rest of the cell membrane. LRs are also referred to as
glycosphingolipid-enriched domains (GEM) or as detergent-resistant membranes (DRM) due to the fact that they are not dissociated after exposure to non-ionic detergent, such as Triton-X 100 or Brij-58, due to their high cholesterol and sphingolipid composition (Simons 1997; Brown 1998). Due to the low density of DRMs, they can be isolated via sucrose gradient density centrifugation, a process which causes DRMs to travel to areas of low sucrose density. lt bas been criticized that DRMs are nonphysiological entities that are generated during the processes of non-ionic detergent extraction and/or density gradient centrifugation. However, high
resolution microscopy evidence using cholera toxin (CTX) B subunit (CTB), a pentamer which binds to significant amounts of the monosialotetrahexosylganglioside (GMl) ganglioside, a
molecule that is heavily enriched in LRs, supports the notion that LRs do exist in physiologically normal intact cells (Bromley 2001; Heerklotz 2002).
Due to their structure, LRs attract certain proteins and exclude others (Simons 1997; Kabouridis 2006). One such protein included in the LR is Lck. lt was found wben comparing detergent-soluble and insoluble lysates using Jurkat T cells tbat approximately 25-50% of total Lck copurified with the DRM fraction (Janes 2000). lt was also determined in two separate studies that Lck association with LRs is critical in connecting TCR-ligation with downstream signaling (Kabouridis 1997; Hawash 2002). LAT is another protein wbicb is localized with LRs and disruption ofthis association disrupts signaling downstream of the ITAM (Zhang 1998; Zhang 1998).
Several reports bave sbown that after TCR activation there is a transient increase in the amount of certain proteins present in LRs. These include: TCRs and ITAMs, ZAP-70, PLCyl, SLP-76, growth factor receptor-bound protein 2 (Grb2) and PKC8 (Montixi 1998; Xavier 1998; Pizzo 2003; Kabouridis 2006). lt has therefore been proposed that upon TCR ligation, TCRs translocate to LRs wbere they may be phosphorylated by LR-resident Lck. LR-associated Lck is innately inactive, due to the activity of the protein associated with GEM (PAG)/Csk-binding protein (Cbp)-Csk inhibitory complex, and mecbanisms underlying the activation of Lck remain unclear. lt is possible, however, that a pbospbatase, namely CD45, may relieve inhibition on Lck (Kabouridis 2006) (Fig. 9).
CD45 G
Csk
~
APC
Tcell
Signal
Figure 9: A model describing the phosphorylation of the ITAM in LR.
QLck
045
ü
In resting T cells, Lck, as well as LAT (not shown), is constitutively associated with LR. Lck forms a weak interaction with the ITAMs and its activity is kept in its basal state due to the activity of the PAG/Cbp-Csk inhibitory complex. After TCR-pMHC ligation, there is an increase in the association of the TCR, as well as Zap-70, PLCy 1, SLP-76, Grb2 and PKC8 (not shown), with LRs. After TCR stimulation there is a transient redistribution of CD45 to these domains, which leads to dephosphorylation ofLck-Y505 and of PAG/Cbp and the dissociation ofCsk
from the PAG/Cbp-Csk complex. This increase in Lck activity results in ITAM phosphorylation and a subsequent signaling cascade [Illustration modified from (Kabouridis 2006)].
1.6. Basal homeostatic proliferation of CDS+ T cells
Thymie output of T lymphocytes is significantly reduced upon reaching puberty. To compensate for this, the T cells present in secondary lymphoid tissues are maintained at consistent levels by slow turnover in a process called homeostatic proliferation. During this process the ratio ofCD4+/CD8+ T cells remains constant (Rocha 1989). Naive and memory cells proliferate independently of each other and in their own niches. This homeostatic proliferation also maintains the proportion and polyclonality of naive and memory CDS+ T cells relatively constant (Tanchot 1998; Surh 2002).
Naïve and memory T cells appear to have different survival requirements in order to maintain their basal homeostatic proliferation. Studies using MHC-deficient mice have shown that naive cells require pMHC:TCR interaction in addition to IL-7 for their survival. Memory T cells, which proliferate in the absence of TCR stimulation, require signals through both the IL-7 and IL-15 receptors for survival. In the presence of abundant IL-7 memory T cells can proliferate without the need for IL-15 (Murali-Krishna 1999; Tan 2001; Schluns 2003; Surh 2005).
1. 7. Acute homeostatic proliferation of CDS+ T cells
T Lymphopenia is a condition in which an organism displays a reduction in T cell numbers in the secondary lymphoid organs. Lymphopenia can be caused by several factors, which allows us to subcategorize three different forms ofT lymphopenia: Transient
lymphopenia, chronic lymhopenia and iatrogenic lymphopenia. Transient lymphopenia occurs in the aftermath of certain viral infections, chronic lymphopenia occurs when there is a steady-state reduction in lymphocytes, such as what is observed in patients infected with HIV, and iatrogenic lymphopenia occurs following radiotherapy and chemotherapy. Elderly people, who have a decreased output of thymie T lymphocytes, may be impacted more severely by T lymphopenia (Jameson 2002).
The process in which T cells expand under conditions ofT lymphopenia is referred to as LIP. LIP occurs in the absence of a functional thymus and involves the division of existing lymphocytes in the secondary lymphoid organs. During LIP the remaining T cells either
proliferate evenly to fill the T cell compartment, or certain T cells have a proliferative advantage and thus proliferate unevenly to fill this niche. The first scenario leads to an overall reduction in diversity in a 'full' compartment, whereas the second scenario leads to an oligoclonality resulting
in an even greater reduction in TCR diversity. This oligoclonality occurs with T cells whose
TCRs are at the limit of the threshold level of activation by self Ags and thus may have a proliferative advantage over others. This oligoclonality could potentially lead to autoimmunity since the T cells with the proliferative advantage possess TCRs that recognize self Ag (Khoruts 2004) (Fig. 10).
As described in the previous section, the basal homeostatic proliferation of T cells is driven by the interaction of the TCRs of these cells with self pMHC and IL-7. LIP is driven by this interaction as well and IL-7 is in abundance under conditions oflymphopenia (Fry 2001; Surh 2005). The rapidity of LIP may cause naive T cells to gain the expression of certain memory markers: CD122hi, CD132h1, CD44hi and Ly6C. These cells do not however gain the
expression of activation markers such as CD25 and CD69 (Cho 2000; Goldrath 2000; Murali-Krishna 2000; Kieper 2002). These cells have a similar phenotype to memory cells following an antigenic response, but do not display effector functions. These cells gain high levels of effector functions, like true memory cells, after stimulation with cognate Ag (Ramanathan 2009).
Steady state Lym phopen
ia-i nducia-i ng ia-insult
~-:-:
•••
oe**
1. Normal T cell population size••
••
~
••
*
f-••• °*
Recovery via LIP where all T cell proliferate equally
1. Normal T cell population size 2. Limited TCR diversity
•
•
•
•*
2. Great TCR diversity.t•·
-+0
**
•*·
*
Recovery via LIP where some T cells have a selective advantage over others
1. Normal T cell population size 2. Oligoclonal expansion
3. Greatly reduced TCR diversity 4. Potential automimmunity Recovery in presence of a functional thymus
Figure 10: Representation of immune cell reconstitution following T lymphopenia. Each T cell is shown as a unique symbol, which represents a unique TCR. Under normal conditions, the T cell repertoire bas vast diversity. Many of these cells are lost following T lymphopenia and the diversity of these cells can be renewed in the presence of a functional thymus. In the absence of a functional thymus the T cell population is restored via LIP, which results in a Joss of TCR diversity. The potential scenarios of equal and unequal proliferation of T cells is illustrated [Illustration modified from (Khoruts 2004)] .
1.8. Antigen-independent proliferation of CDS+ T cells induced by cytokines leads to a state of 'cytokine priming'
It has been shown that naive CD441°CDS+ T cells are able to undergo antigen
non-specific proliferation in the presence of a homeostatic cytokine, such as IL-7 and IL-15, in combination with an inflammatory cytokine, such as IL-6 or IL-21 (Fig. 11) (Zeng 2005; Gagnon 2007). Furthermore, naive CDS+ T cells that have been stimulated with these synergistic
combinations of cytokines develop increased reactivity towards cognate antigens. This bas been shown using CDS+ T cells expressing transgenic TCRs, particularly, those expressing the P14 TCR, which specifically recognizes a peptide spanning amino acids 33-41 of the glycoprotein (GP) antigen oflymphocytic cboreomeningitis virus (LCMV) (Gagnon 200S). Specifically, pre-incubation of P14 TCR transgenic CDS+ T cells with IL-7 or IL-15 in the presence ofIL-6 or IL-21 leads to proliferation, increased pbosphorylation and DNA binding of signal transducer and activator of transcription 5 (STAT5) and modulation of the expression of several cell surface molecules such as the increase in CDS, CD45 and costimulatory molecules that can positively regulate TCR signaling, and decrease in CD5 that exerts a negatively regulatory effect on TCR signaling (Gagnon 200S; Gagnon 2010). Furthermore, it was found that stimulation with the combination ofIL-7 and IL-21for48 hours leads to increased LAT-Yl 71 phosphorylation after CD3 cross-linking (Gagnon, Unpublisbed Data). As a consequence, these cytokine-prestimulated cells display increased proliferation and effector functions upon subsequent Ag stimulation as indicated by increased secretion ofIL-2 and IFNy, expression of granzyme B and more efficient Ag-specific cytotoxicity towards target cells (Gagnon 200S; Gagnon 2010).
The above findings gave raise to the concept of 'cytokine priming' of naïve CDS+ T cells.
It has been proposed that naïve CDS+ T cells exposed to the stimulatory combinations of IL-15 and IL-21, a situation that could occur during inflammation caused by innate immune response, could sensitizes these cells to lower concentrations of Ag (Ramanathan 200S) (Fig. 12). These 'primed' cells proliferate in response to lower concentrations of Ag and have more potent
effector functions. It has been shown that cytokine-primed cells can up-regulate the IL-2 receptor (IL-2R) a and~ as well as CD62L and CD44, which might promote their continued migration in search of Ag (Gagnon 200S). Therefore it is possible that during an immune response, CDS+ T cells are recruited to the site of inflammation where they are stimulated by inflammatory cytokines derived from the innate immune system. This may sensitizes these cells to become more responsive to invading pathogens (Ramanathan 2009).
A
IL-7 CTLs Pathogens1 1
1 1
lnnate immune stimulation
Clonai expansion Memory cell Inflammation Neutrophils Macrophages Dendritic cells
Mast cells Transient Fibroblasts lymphopenia NKT cells
I
@Naive~
IL-15 ....+-
IL-7 IL-61
IL-21~
::::::::::
- Partial effector1
functionsfN
Memory-like - lncreased Ag ~ responsiveness Potential implications:- Augmentation of antigen-specific response (transition from innate to adaptive immune response)
- Triggering autoreactive CDS T cells - Useful to expand tumor-specific CTLs
Figure 11 _ Ag-independent activation of naïve CD8+ T cells by cytokines.
(A) Activation of CD8+ T cells traditionally occurs following pMHC:TCR ligation between APCs and CD8+ T cells. Stimulated cells proliferate with the help of autocrine IL-2.
B
Furthermore, the impact of various inflammatory cytokines are necessary to maxirnize the CD8+ T cell response. Most of these cells become effector cells and die upon elirnination of the Ag source. Sorne of the activated cells differentiate into memory cells with the help of IL-7 and are maintained by IL-15 to provide long-term immunity. (B) Under inflammatory conditions, IL-15, IL-21 and IL-6 are produced by cells of the innate immune system. These cytokines can
stimulate naïve CD8+ T cells and activate them in an Ag-independent manner. This could increase in the CD8+ T cell responsiveness to Ag that may augment the adaptive immune
responses which could be both useful in eradicating infected cells, but may also be detrimental as this may also trigger potentially autoreactive CDS+ T cells [Illustration modified from
Na ive CD8+ Tcell or
---:~IL-~7~+~1L~-6--).,
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or IL-7 + IL-6 Proliferation IL-2, IFNy secretionAg-specific cytolytic activity
Significant proliferation No IL-2 or IFNy secretion
Negligible Ag-specific cytolytic activity
Limiting
_a_nti-ge_n -)..
©
1 High antigen sensitivity 1
lncreased proliferation lncreased transcription of
112 and lfng genes lncreased secretion of
IL-2 and IFNy lncreased expression of
granzyme B Elevated Ag-specific
cytolytic activity
Figure 12. The concept of 'cytokine Priming' of naive CD8+ T cells.
Cytokine synergy induces Ag-independent proliferation of naive CD8+ T cells and primes them to display potent effector functions upon subsequent Ag stimulation. During an immune
response, cells of the innate immune system produce IL-15, IL-21, and IL-6. Meanwhile, IL-7 is continuously available. The synergistic combination of IL-7 with IL-21 or IL-6 can stimulate proliferation of naive CD8+ T cells in the absence of its cognate Ag. These 'cytokine-primed' CD8+ T cells possess a memory-like phenotype and are more sensitive to low concentrations of cognate Ags. Upon stimulation with this Ag, the cytokine-primed CD8+ T cells secrete abundant quantities ofIL-2 and IFNy, and have more potentAg-specific cytolytic activity on target cells [Illustration modified from (Ramanathan 2009)].
